The proposed work focuses on basic structure/activity studies of two molybdenum-containing enzymes, xanthine oxidoreductase and carbon monoxide dehydrogenase. The first enzyme catalyzes the final two steps in purine metabolism in humans, and is also an important therapeutic target in the treatment of hyperuricemia and also in chemotherapeutic regimens. It has become the paradigm for mechanistic studies of the molybdenum hydroxylase family of enzymes. The second enzyme catalyzes the key step in carboxydotrophic growth on CO in certain bacteria and is responsible for removal of approximately 2 x 108 metric tons of CO from the environment each year. It is also a biological reaction of fundamental interest from a chemical standpoint: although CO dehydrogenase exhibits significant structural and sequence homologies to xanthine oxidoreductase, it is unique among the molybdenum hydroxylases in that its reaction does not involve C-H bond cleavage. The overall goal of the proposed work is to gain a more complete understanding of the mechanism of action of these two enzymes in the context of their structures, the guiding hypothesis being that enzyme function is dictated by the physical and electronic structure of the active site. The Specific Aims include rapid kinetic studies as well as spectroscopic work aimed at determining the electronic structures of intermediates identified. In the case of xanthine oxidoreductase, site-directed mutants targetting specific active site amino acid residues will be examined to evaluate their roles in catalysis. The overall goal is to compare and contrast the behavior of these two closely related enzymes so that the relationship of structure to function can be better understood.